Although DNA vaccines were developed more than 16 years ago, clinical trials preceding stage I and II in humans are rare. Three veterinary DNA vaccines however, have been licensed (Kurtzler M A & Weiner D B. DNA vaccines: ready for prime time? Nature rev 2008); one for West Nile Virus (in horse), one for Infectious Hematopoetic Necrosis virus in Salmon, one against Melanoma in dogs, and a plasmid for growth hormone releasing hormone in swine. This demonstrates that DNA vaccines/therapies can have good protective effects and that new DNA vaccines are not limited by the size of the animal or species. The great success with DNA vaccines observed for the murine model for first generation DNA vaccines did not translate well to humans, nonetheless; researchers have recently demonstrated protective antibodies levels by a single dose of gene gun administrated HA DNA vaccine to humans. Therefore, there is a future for improved DNA vaccines in human and veterinarian clinics.
“Nucleic acid immunization” or the commonly preferred name “DNA vaccines” are the inoculation of antigen-encoding DNA or RNA as expression cassettes or expression vectors, which may also be incorporated into viral delivery vectors with the purpose of inducing immunity to the gene product. Thus, in our definition of DNA vaccines we include all kinds of delivery systems for the antigen encoding naked DNA or RNA including viral vector-based delivery. The vaccine genes can be in form of circular plasmid or a linear expression cassette with just the key features necessary for expression (promotor, the vaccine gene and polyadenylation signal). Delivery systems may most often be naked DNA in buffer with or without adjuvant, DNA coupled to nanoparticles and/or formulated into adjuvant containing compounds or inserted into live viral or bacterial vectors such as Adenovirus, adeno-associated virus, alphavirus, poxviruses, herpes virus etc.
DNA vaccines hold great promise since they evoke both humoral and cell-mediated immunity, without the same dangers associated with live attenuated virus vaccines. In contrast to live attenuated virus vaccines DNA vaccines may be delivered to the same or different tissue or cells than the live virus that has to bind to specific receptors. The production of antigens in their native forms improves the presentation of the antigens to the host immune system. Unlike live attenuated vaccines, DNA vaccines are not infectious and cannot revert to virulence. DNA vaccines expressing influenza virus HA, NA, M, NP proteins or combinations of these have proven to induce immune responses comparable to that of a natural viral infection.
DNA vaccines offer many advantages over conventional vaccines. It can be produced in high amounts in short time, abolishing the need for propagation in eggs, it is cost-effective, reproducible and the final product does not require cold storage conditions, because DNA is stable and resistant to the extremes of temperature. All currently licensed inactivated vaccines are efficient at inducing humoral antibody responses but in general only live attenuated virus vaccines and DNA vaccines efficiently induce a cytotoxic cellular response as well. Moreover, The DNA vaccine's protein product is produced in vivo by the host cells believed to ensure more correct folding and glycosylation and presentation to the immune system than in vitro produced protein or glycoprotein immunogens.
DNA vaccines induce an immune response, which is comparable to the response acquired by natural virus infection by activating both humoral immunity to also native proteins or glycoproteins as well as cell-mediated immunity to intracellular processed immunogens (6, 30). The broad response to DNA vaccines is a result of the encoded genes being expressed by the transfected host cell, inducing both a Th1 and Th2 immune responses. The production of antigens in their native form improves the presentation of the antigens to the host immune system.
The standard DNA vaccine consist of a vector backbone with the gene of interest cloned into a bacterial plasmid engineered for optimal expression in eukaryotic cells. Essential features include; an origin of replication allowing for production in bacteria, a bacterial antibiotic resistance gene allowing for plasmid selection in bacterial culture during production of plasmid DNA, a strong constitutive promotor for optimal expression in mammalian cells (e.g. promoters derived from cytomegalovirus (CMV) or simian virus provide the highest gene expression), a polyadenylation sequence to stabilise the mRNA transcripts, such as bovine growth hormone (BHG) or simian virus polyadenylation signals, and a multiple cloning site for insertion of an antigen gene.
An intron A sequence improves expression of genes remarkably and may be included in the expression plasmid backbone. Many bacterial DNA vaccine vectors contain unmethylated cytidinephosphate-guanosine (CpG) dinucleotide motifs that may help eliciting adjuvanting innate immune responses in the host. In recent years, there have been several approaches to enhance and customise the immune response to DNA vaccine constructs (2nd generation DNA vaccines). For instance, dicistronic vectors or multiple gene expressing plasmids have been used to express two genes simultaneously. Specific promoters have been engineered that restrict gene expression to certain tissues, and cytokine/antigen fusion genes have been constructed to enhance the immune response. Furthermore, genes may be codon optimised for optimal gene expression in the mammalian host and naïve leader sequences may be substituted with stronger optimised leaders increasing translation efficiency.
The two most common types of naked DNA vaccine administration have so far been saline or PBS (phosphate buffered saline) needle injection of naked DNA and gene gun DNA inoculations (DNA coated on solid gold beads administrated with helium pressure). A saline intra muscular injection of DNA preferentially generates a Th1 IgG2a response while gene gun delivery tends to initiate a more Th2 IgG1 response. Intramuscular injected plasmids are at risk of being degraded by extracellular deoxyribonucleases, however, the responses induced are often more long-lived than those induced by the gene gun method. Vaccination by gene gun delivery of DNA, to the epidermis, was considered the most effective method of immunization, probably because the skin is a very immunogenic organ containing all the necessary cells types, including professional antigen presenting cells (APC), for eliciting both humoral and cytotoxic cellular immune responses (e.g. Langerhans and dendritic cells).
In 2003 electroporation of DNA was introduced as a way of improving transfection of mammalian cells with foreign DNA e.g. in cancer treatment. It is now a well recognized way of administration a naked DNA vaccine and one of the most efficient and most potent delivery methods so far. Electroporation, or electropermeabilization, is caused by an externally applied electrical field, which results in a significant increase in the electrical conductivity and permeability of the cell plasma membrane thereby subsequently transferring the administered DNA into the cells in the administered area. The organ targeted for the delivery with electroporation is most often muscle and/or skin. The disadvantages or challenges using needle and using electroporation is several. Intradermal injection require skills to perform and the vaccine is only deposited at the site where the needle tip ends up. In contrast, a needle-free jet delivery targets several layers of the skin reaching APC in different layers and do not require the same expert skills. In addition, needle's possess a risk for needle injury to the handler. More over the relatively time consuming process of both injection and electroporation may require immobilization of the animal and possible anaesthesia, access to electricity etc. Even with hand held cordless devices the technique delivers an electric pulse that may be painful. Because of the very efficient transfection obtained by moving plasmid DNA by the electric field during electroporation there is a fear that such foreign plasmid DNA may have increased possibility of integrating into the host chromosomes. This would become a safety risk. In contrast, a method that delivers the DNA vaccine fast without the need for needles and electroporation would be a considerable advantage and be an improvement in animal health and safety for the person who vaccinates. However, until now the most efficient mode of deliver DNA vaccine for optimal immune induction has been injection either intramuscular or intradermal followed by electroporation. The use of adjuvants is most wanted for DNA vaccines; however, such attempts has been mostly by codelivery of adjuvanting compounds such ad cytokines or plasmids encoding cytokines e.g. GM-CSF, IL-15.